CN110484455A - A kind of trichoderma mutant strain of stable, high-yielding phytase - Google Patents
A kind of trichoderma mutant strain of stable, high-yielding phytase Download PDFInfo
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- CN110484455A CN110484455A CN201910494545.5A CN201910494545A CN110484455A CN 110484455 A CN110484455 A CN 110484455A CN 201910494545 A CN201910494545 A CN 201910494545A CN 110484455 A CN110484455 A CN 110484455A
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Abstract
The present invention provides a kind of trichoderma mutant strain of high phytase generating and its applications.The deposit number of the mutant strain is CCTCC NO:M2019405, and the enzyme activity of phytase reaches 3580u/mL in shake flask fermentation supernatant, improves 56.0% than going out bacterium germination；Phytase activity is up to 40345u/mL in 20L tank fermented supernatant fluid, improves 52.1% than starting strain, unexpected technical results have been achieved.The application of the mutant strain can further decrease the production cost of phytase, be conducive to accelerate extensive use of the phytase in field of fodder.
Description
Technical field
The invention belongs to microbial project renovation technique fields, and in particular to a kind of trichoderma mutation of stable, high-yielding phytase
Bacterial strain and its application.
Background technique
Phytic acid (phytic acid), also known as phytic acid (myo-inositol(1,2,3,4,5,6)
It hexakisphosphate), is the principal mode that phosphorus is stored in plant, content is especially abundant in seed, and seed such as cereal
And beans is the primary raw material of animal feed.Although the important sources of phosphorus needed for the phytic acid in seed can become raising animal,
Only ruminant ability energy metabolism phytic acid is to utilize phosphorus therein；For non-ruminant animal, the phytic acid that cannot be digested metabolism is anti-
And it is considered as anti-nutrient substance.And it is phytic acid with negative electricity abundant that phytic acid, which is considered as the reason of anti-nutrient substance, easily with have
The ion of positive electricity, if calcium ion, magnesium ion, zinc ion, manganese ion, copper ion, iron ion chelating, further with protein and
Starch formation compound, this compound not only hinder the digestion and absorption of metal ion, also influence digestion enzyme effect and hinder nutrition
Material absorbing.
Phytase (Phytase) is to be catalyzed phytic acid and phytate hydrolysis into one kind of inositol and phosphoric acid (or phosphate)
The general name of enzyme.Phytase has special space structure, can successively separate the phosphorus in phytate molecule, phytate is degraded to nothing
Machine phosphorus and inositol, while releasing other nutriments of phytate combination.Currently, utilizing the effect of phytase feeding nonruminant
Verifying has been obtained in fruit.After adding phytase in feed, it is possible to reduce the dosage of 5-70% Phos, the discharge of phosphorus in excrement
Amount reduces 30-40% or more, not only greatly reduces the anti-oxidant action of phytate, increases productivity effect, moreover it is possible to effective drop
Low environment pollution.Phytase has been widely used in livestock and poultry cultivation field as feed addictive.
Since the yield of phytase in natural bacterial strain is lower, the phytase that natural bacterial strain produces in addition is in thermal stability, albumen
The requirement of feed processing and phytase application cannot be fully met in terms of the zymologic properties such as enzyme resistance.Therefore, those skilled in the art
Member generallys use technique for gene engineering means and is transformed to wild type phytase to obtain the mutant protein of function admirable, structure
Pichia pastoris, aspergillus niger are built, the engineering strains such as aspergillus oryzae are used for the fermenting and producing of phytase, can increase substantially phytase
Enzyme activity it is horizontal, may advantageously facilitate the extensive use of phytase.Currently, the unit expression quantity of phytase production bacterial strain is improved, into
It is still one of most important phytase industry goal that one step, which reduces its production cost,.
Summary of the invention
The present invention is to solve prior art problem, provides the Li's Trichoderma strains of one plant of stable, high-yielding phytase and its answers
With.The bacterial strain can increase substantially the expression quantity of phytase, be conducive to the extensive use of phytase.
In order to achieve the above-mentioned object of the invention, the invention provides the following technical scheme:
One aspect of the present invention provides a kind of trichoderma reesei engineering bacteria, carries the recombinant vector of Expressing Recombinant Phytase gene.
The phytase, amino acid sequence are SEQ ID NO:1, and the sequence of encoding gene is SEQ ID NO:2.
The present invention also provides a kind of mutant strain trichoderma reesei UEphy-6(Trichoderma reesei UEphy-
6) China typical culture collection center of Wuhan, China Wuhan University, deposit number, are preserved on May 29th, 2019
For CCTCC NO:M2019405.
The present invention also provides application of the mutant strain in phytase production.
The mutant strain trichoderma reesei UEphy-6 that applicant is obtained by mostly wheel mutagenesis screening, phytic acid production of enzyme obtain
It is obviously improved.The enzyme activity of phytase reaches 3580u/ml in trichoderma reesei UEphy-6 shake flask fermentation supernatant, improves than going out bacterium germination
56.0%；Phytase activity is up to 40345u/ml in 20L tank fermented supernatant fluid, improves 52.1% than starting strain, achieves
Unexpected technical effect.The application of the mutant strain can further decrease the production cost of phytase, be conducive to accelerate
Extensive use of the phytase in field of fodder.
Detailed description of the invention
Fig. 1 is 20L ferment tank curve；
Fig. 2 is SDS-PAGE protein electrophoresis figure: wherein: M is molecular weight of albumen Marker, and swimming lane 1,2 is respectively trichoderma reesei
UEphy-P2, trichoderma reesei UEphy-6 fermented supernatant fluid are phytase Phy at arrow meaning.
Specific embodiment
The routine techniques and method that the present invention has used genetic engineering and molecular biology field uses, such as
MOLECULAR CLONING:A LABORATORY MANUAL, 3nd Ed. (Sambrook, 2001) and CURRENT
Documented method in PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, 2003).These generality are with reference to text
It offers and provides definition well known by persons skilled in the art and method.But the present invention is not limited to any specific method,
Experimental program and reagent.
Below with reference to specific embodiment, the present invention will be described in detail.
Embodiment 1: the clone of phytase gene and the building of expression vector
According to trichoderma (Trichoderma sp.) codon preference, by Escherichia coli (Escherichia coli) source
The amino acid sequence SED ID NO:1 of phytase Phy gene carried out codon optimization, had by general biosystem (Anhui)
Limit company synthesizes its coding nucleotide sequence SED ID NO:2.
Upstream and downstream primer Phy-F and Phy-R are designed according to the nucleotide sequence of synthesis, sequence is as follows:
Phy-F:GGCTCTAGACAGTCGGAGCCCGAGCTGAAGC；
Phy-R:ATAACGCGTTTAGAGCGAGCAGGCGGGAATT。
It is arranged with the nucleotides sequence of synthesis as template, is expanded using upstream and downstream primer Phy-F and Phy-R, utilize gel
QIAquick Gel Extraction Kit recycles pcr amplification product.Above-mentioned pcr amplification product is subjected to double enzymes with restriction enzyme XbaI and MluI
It cuts, while with XbaI and MluI double digestion expression vector pC2G and pC1G, by pcr amplification product double digestion segment and expression vector
The connection of pC2G double enzyme digestion product overnight, imports escherichia coli DH5a, after sequence verification, obtains recombinant expression carrier pC2G-
Pcr amplification product double digestion segment connect overnight with expression vector pC1G double enzyme digestion product, imports escherichia coli DH5a by Phy,
After sequence verification, recombinant expression carrier pC1G-Phy is obtained.
Embodiment 2: the building of the trichoderma reesei engineering bacteria UEphy of primary conversion phytase gene
1, prepared by protoplast
Take trichoderma reesei (Trichoderma reesei) UE bacterial strain spore suspension, it is inoculated on PDA plate, 30 DEG C of cultures 6
It；After its produce spore it is abundant after, cut about 1cm × 1cm bacterium colony be placed in containing 120 mL YEG+U(0.5% yeast powders, 1% glucose,
0.1% uridine) fluid nutrient medium in, 30 DEG C, 220 rpm shaken cultivation, 14 ~ 16 h；
Mycelium is collected by filtration with sterile gauze, and primary with sterile water wash；Mycelium is placed in containing 20 mL 10mg/mL
In the triangular flask for cracking enzyme solution (Sigma L1412), 30 DEG C, 90 rpm act on 1-2 h；Protoplast is detected with micro- sem observation
Conversion progress；
By 20 mL, 1.2 M sorbierite (1.2 M sorbierites, the 50 mM Tris-Cl, 50 mM CaCl of pre-cooling2) be added it is above-mentioned
It in triangular flask, gently shakes up, collects filtrate, 3000 rpm, 4 DEG C of 10 min of centrifugation with sterile Miracloth filter-cloth filtering；In abandoning
Clearly, 5 mL, the 1.2 M sorbitol solution suspension thalline of pre-cooling, 3000 rpm, 4 DEG C of 10 min of centrifugation are added；Supernatant is abandoned, is added
1.2 M sorbierites being pre-chilled in right amount suspend, and (200 μ L/ are managed, protoplast concentration 10 for packing8A/mL).
2, expression vector converts
It operates and carries out on ice below, 10 μ g recombinant plasmid pC2G-Phy is taken to be added to containing 200 μ L protoplast solutions
7 mL sterile centrifugation tubes in, 50 μ L 25% PEG(25% PEG, 50 mM Tris-Cl, 50 mM CaCl is then added2),
Tube bottom mixing is flicked, places 20 min on ice；2 mL, 25% PEG is added, 5 min are placed at room temperature for after mixing；4 mL 1.2 are added
M sorbierite pours into (0.1%MgSO in the upper layer culture medium for melt and be maintained at 55 DEG C after mixing gently4, 1%KH2PO4, 0.6%
(NH4)2SO4, 1% glucose, 18.3% sorbierite, 0.35% agarose)；The lower layer's culture prepared is layered on after mixing gently
(2% glucose, 0.5% (NH on base plate4)2SO4, 1.5%KH2PO4, 0.06%MgSO4, 0.06%CaCl2, 1.5% agar), 30 DEG C
5 ~ 7 d are to there is transformant to grow for culture, and the transformant grown is chosen to lower layer's culture medium flat plate secondary screening, colony edge form compared with
Smooth bacterial strain is positive transformant.
3, fermentation verifying and enzyme activity determination
The positive transformant that above-mentioned secondary screening obtains is seeded to PDA solid plate, in 30 DEG C of constant temperature incubation carton upside down culture 6-7
It, after spore is abundant, take respectively the inoculated by hypha block of two pieces of diameter 1cm in containing 50mL fermentation medium (1.5% glucose,
1.7% lactose, 2.5% corn pulp, 0.44% (NH4)2SO4, 0.09%MgSO4, 2%KH2PO4, 0.04%CaCl2, 0.018% tween-
80,0.018% microelement) 250mL triangular flask in, 30 DEG C are cultivated 48 hours, are then cultivated 48 hours for 25 DEG C, are taken in fermentation
Clear liquid carries out the test of phytase activity power.
(1) enzyme activity determination method
Enzyme activity definition: under the conditions of 37 DEG C of temperature, pH5.5,1 μ is discharged from concentration 5.0mmol/L sodium phytate solution per minute
Mol/L Phos, as a phytase activity unit, are indicated with U.
Measuring method: it accurately weighs the benchmark potassium dihydrogen phosphate (5.9) that 0.6804g dries to constant weight at 105 DEG C and holds in 100ml
In measuring bottle, dissolved with acetate buffer (5.1), and be settled to 100ml, concentration 50.0mmol/L.In the ratio acetic acid of table 1
Buffer (5.2) is diluted to various concentration, the reaction assay together with sample to be tested.Using inorganic phosphorus concentration as abscissa, light absorption value
For ordinate, linear regression equation (y=ax+b) is listed.
Sample after reaction stands 10min in a water bath, is centrifuged 10min on centrifuge (6.7) with 4000r/min, on
Clear liquid is returned to zero with the blank of standard curve, and sample blank (A is measured at spectrophotometer (6.3) 415nm wavelength0) and sample it is molten
The light absorption value of liquid (A), A-A0To survey light absorption value.The activity of phytase is calculated with linear regression equation.
Phytase activity is calculated as follows:
U=F×C/(m×30)
In formula:
The activity of phytase, U/g in U-- sample；
C-- is according to the light absorption value of practical sample liquid by the calculated enzymatic activity of linear regression equation, U；
Total extension rate before the reaction of F-- sample solution；
M-- sample mass, g；
30-- reaction time, min.
The results show that the trichoderma reesei engineering bacteria shake flask fermentation supernatant enzyme activity that the present invention constructs can reach
1970U/ml.Applicant's highest this plant of trichoderma reesei engineering bacteria of enzyme activity that will ferment is named as trichoderma reesei UEphy
(Trichoderma reeseiUEphy).
The building of the trichoderma reesei engineering bacteria UEphy-P2 of 3 secondary conversion phytase gene of embodiment
1, the preparation of uracil-deficient host strain
1.1 principles:
5- fluororotic acid can induce orotidine monophosphate transferase or whey in thallus missing uridylate route of synthesis
Glycosides monophosphate decarboxylase, to make 5- fluororotic acid that can not form toxic substance 5 FU 5 fluorouracil nucleotide, to produce
To the resistance of 5- fluororotic acid, pyrimidine nucleotide nutrition can be supplemented by adding uracil into culture medium, therefore
It can be in the training containing 5- fluororotic acid and uracil using the uracil auxotrophy bacterial strain of 5- fluororotic acid induced synthesis
It supports and is grown in base；And wild-type strain is because having the resistance to 5- fluororotic acid, it can not be in the culture containing 5- fluororotic acid
Under the conditions of grow.Therefore 5- fluororotic acid is commonly used to screen the mutant strain of uracil-deficient.
1.2 screening techniques:
By the trichoderma reesei UEphy(of fresh collectionTrichoderma reeseiUEphy spore) is with 0.1% Tween-20
Solution is diluted to about 1 × 107A/ml is coated on the fluororotic acid of 5- containing 1.5g/ml and 1.87g/ml Uridine(uridine diphosphate
Glycosides) basic solid medium (2% glucose, 0.5% (NH4)2SO4, 1.5%KH2PO4, 0.06%MgSO4, 0.06%CaCl2,
1.5% agar) plate, each plate coating about 1 × 106A spore is protected from light 30 DEG C of culture 4d；By what is grown in above-mentioned plate
Bacterial strain is respectively connected to minimal medium plate and the minimal medium plate containing 1.87mg/ml Uridine, is only containing
Grown on the plate of Uridine and without Uridine the non-growing bacterial strain of minimal medium plate be uracil-deficient
Mutant strain is named as trichoderma reesei UEphy-P(Trichoderma reeseiUEphy-P).
2, prepared by protoplast
Method is such as embodiment 2.
3, expression vector converts
It operates and carries out on ice below, take 10 μ g recombinant plasmid pC1G-Phy to be added to molten containing 200 μ L protoplasts
In the 7mL sterile centrifugation tube of liquid, 50 μ L, 25% PEG (25% PEG, 50mM Tris-Cl, 50mM CaCl2) is then added,
Tube bottom mixing is flicked, places 20min on ice；25% PEG of 2mL is added, is placed at room temperature for 5min after mixing；4mL is added
1.2M sorbierite, poured into after mixing gently in the upper layer culture medium for melting and being maintained at 55 DEG C (0.1% MgSO4,1%
KH2PO4,0.6% (NH4) 2SO4,1% glucose, 18.3% sorbierite, 0.35% agarose)；It is spread after mixing gently
(2% glucose, 0.5% (NH4) 2SO4,1.5% KH2PO4,0.06% on the lower layer's culture medium flat plate prepared
MgSO4,0.06% CaCl2,1.5% agar), 30 DEG C of 5 ~ 7d of culture are to there is transformant to grow.
The transformant grown is chosen to lower layer's culture medium flat plate and is screened again, the more smooth bacterium of colony edge form is chosen
Strain is forwarded to PDA plate and is cultivated.
4, fermentation verifying and enzyme activity determination
The positive transformant that above-mentioned secondary screening obtains is seeded to PDA solid plate, in 30 DEG C of constant temperature incubation carton upside down culture 6-7
It, after spore is abundant, take respectively the inoculated by hypha block of two pieces of diameter 1cm in containing 50mL fermentation medium (1.5% glucose,
1.7% lactose, 2.5% corn pulp, 0.44% (NH4)2SO4, 0.09%MgSO4, 2%KH2PO4, 0.04%CaCl2, 0.018% tween-
80,0.018% microelement) 250mL triangular flask in, 30 DEG C are cultivated 48 hours, are then cultivated 48 hours for 25 DEG C, are taken in fermentation
Clear liquid carries out enzyme activity determination, and enzyme activity determination method is shown in embodiment 2.
The results show that the trichoderma reesei engineered strain fermentation enzyme activity that secondary conversion of the present invention obtains can reach 2295U/
Ml, applicant's highest this plant of trichoderma reesei engineering bacteria of enzyme activity that will ferment are named as trichoderma reesei UEphy-P2(Trichoderma reeseiUEphy-P2).
4 ultraviolet mutagenesis of embodiment and screening
Mutation randomness caused by ultraviolet mutagenesis is very strong, and it is also random for being mutated the effect of generation, it is difficult to be predicted.Therefore, in order to
Effective direct mutation is obtained, technical staff usually requires to carry out more wheel ultraviolet mutagenesis, the larger workload of screening, and there are nothings
Method obtains a possibility that effective direct mutation.But it because equipment needed for ultraviolet mutagenesis is simple, expense is few, and can obtain in a short time
Mass mutation body is obtained, therefore, it is still a kind of common mutagenic breeding method now.
Applicant carries out science of heredity to it by ultraviolet mutagenesis method and changes using trichoderma reesei UEphy-P2 as starting strain
It makes, further increases the yield of its phytase.
1, lethality is determined:
Trichoderma reesei engineering bacteria UEphy-P2 is inoculated in PDA plate, 30 DEG C of culture 5-7d.A large amount of spores are generated to bacterium colony surface
When, the sterile water elution of 5ml is drawn, spore liquid is obtained, is resuspended after centrifugation with sterile water, is counted with blood counting chamber.Take one
90mm culture dish, the spore suspension that 5ml has diluted is added, and (concentration is 1 × 107), rotor is added and is stirred on magnetic stirring apparatus
Spore liquid is set to be in uniform state.In aseptic superclean bench, the ultraviolet lamp for being 9w with power is in the upper of vertical range 20cm
Side's irradiation, irradiates 30s, 45s, 60s, 75s, 90s, 105s, 120s respectively, the spore liquid dilution 10,100,1000 after taking irradiation
Times, it takes 100ul to be coated with PDA plate, is counted after 30 DEG C of culture 2-3d, be control with non-irradiated spore liquid, calculate lethality.Its
When middle irradiation 90s, lethality 95% chooses the irradiation time and carries out subsequent Mutagenesis experiments.
2, first round mutagenesis screening:
A 90mm culture dish is taken, the spore suspension that 5ml has diluted is added, and (concentration is 1 × 107), rotor is added and is stirred in magnetic force
Mixing stirring on device makes spore liquid be in uniform state.In aseptic superclean bench, with power be 9w ultraviolet lamp in vertically away from
Top irradiation from 20cm, dilutes 1000 times after irradiating 90s, 100ul is taken to be coated with PDA plate, 30 DEG C of culture 2-3d.
It is coated with 200 pieces of PDA plates altogether, after 30 DEG C of culture 2-3d, each plate grows 30-50 bacterium colony, first passes through bacterium colony
Form filters out the mutant of short branch.Applicant's picking goes out that colonial morphology is smaller, mycelia is fine and close, periphery of bacterial colonies villus is shorter
Mutant bacteria arrive PDA plate, 30 DEG C of culture 5-7d respectively for totally 85.Each transformant extracts the fungus block of 2cm × 2cm size, point
It is not inoculated in 50ml liquid submerged culture base and ferments, 28 DEG C of culture 5d.After cultivating 5d, it is as thick that centrifugation thallus obtains supernatant
Enzyme solution carries out phytase activity detection respectively, while as a control group with starting strain trichoderma reesei engineering bacteria UEphy-P2.
The results show that in the 85 plant mutant bacterium that first round Uv-induced screening obtains, without a plant mutant bacterium fermentation supernatant
The enzyme activity of phytase is higher than bacterium germination in liquid enzyme；Wherein, the enzyme activity and bacterium germination out of 62 plant mutant bacterium are substantially suitable, remaining 23 plants prominent
The enzyme activity for becoming bacterium even generally reduces 8-13% than going out bacterium germination.
Applicant has continued 9 wheel mutagenesis screenings according to the method described above, finally obtains 3 plants of phytic acid production of enzyme and is significantly higher than
The mutant strain of bacterium germination out is respectively designated as trichoderma reesei UEphy-3, UEphy-4, UEphy-6.Wherein, trichoderma reesei
The enzyme activity highest of phytase in UEphy-6 shake flask fermentation supernatant reaches 3580u/ml, improves 56.0% than going out bacterium germination.
Further, applicant is by starting strain trichoderma reesei UEphy-P2 and above-mentioned mutant strain trichoderma reesei UEphy-
6 ferment in 20L tank respectively, and for fermentation diagram as shown in Figure 1, after fermentation 160h, it is as thick that centrifugation thallus obtains supernatant
Enzyme solution carries out protein electrophoresis detection and phytase activity detection respectively.
Electrophoresis detection result illustrates trichoderma reesei UEphy-P2 and Richter scale as shown in Fig. 2, be phytase at arrow meaning
Trichoderma UEphy-6 can effective expression phytase Phy.Enzyme activity assay is the results show that starting strain trichoderma reesei UEphy-P2 is sent out
Phytase activity is 26530u/ml in ferment supernatant, and phytase in the fermented supernatant fluid of mutant strain trichoderma reesei UEphy-6
Enzyme activity is up to 40345u/ml, improves 52.1% than starting strain, unexpected technical results have been achieved.
Applicant is on May 29th, 2019 by trichoderma reesei UEphy-6(Trichoderma reeseiUEphy-6)
It is preserved in the China typical culture collection center of Wuhan, China Wuhan University, deposit number is CCTCC NO:M2019405.
Sequence table
<110>Qingdao Weilan Biology Group Co., Ltd.
<120>a kind of trichoderma mutant strain of stable, high-yielding phytase
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 410
<212> PRT
<213>Escherichia coli (Escherichia coli)
<400> 1
Gln Ser Glu Pro Glu Leu Lys Leu Glu Ser Val Val Ile Val Ser Arg
1 5 10 15
His Gly Val Arg Ala Pro Thr Lys Ala Thr Gln Leu Met Gln Asp Val
20 25 30
Thr Pro Asp Ala Trp Pro Thr Trp Pro Val Lys Leu Gly Trp Leu Thr
35 40 45
Pro Arg Gly Gly Glu Leu Ile Ala Tyr Leu Gly His Tyr Gln Arg Gln
50 55 60
Arg Leu Val Ala Asp Gly Leu Leu Ala Lys Lys Gly Cys Pro Gln Pro
65 70 75 80
Gly Gln Val Ala Ile Ile Ala Asp Val Asp Glu Arg Thr Arg Lys Thr
85 90 95
Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro Asp Cys Ala Ile Thr Val
100 105 110
His Thr Gln Ala Asp Thr Ser Ser Pro Asp Pro Leu Phe Asn Pro Leu
115 120 125
Lys Thr Gly Val Cys Gln Leu Asp Asn Ala Asn Val Thr Asp Ala Ile
130 135 140
Leu Ser Arg Ala Gly Gly Ser Ile Ala Asp Phe Thr Gly His Arg Gln
145 150 155 160
Thr Ala Phe Arg Glu Leu Glu Arg Val Leu Asn Phe Pro Gln Ser Asn
165 170 175
Leu Cys Leu Asn Arg Glu Lys Gln Asp Glu Ser Cys Ser Leu Thr Gln
180 185 190
Ala Leu Pro Ser Glu Leu Lys Val Ser Ala Asp Asn Val Ser Leu Thr
195 200 205
Gly Ala Val Ser Leu Ala Ser Met Leu Thr Glu Ile Phe Leu Leu Gln
210 215 220
Gln Ala Gln Gly Met Pro Glu Pro Gly Trp Gly Arg Ile Thr Asp Ser
225 230 235 240
His Gln Trp Asn Thr Leu Leu Ser Leu His Asn Ala Gln Phe Tyr Leu
245 250 255
Leu Gln Arg Thr Pro Glu Val Ala Arg Ser Arg Ala Thr Pro Leu Leu
260 265 270
Asp Leu Ile Met Ala Ala Leu Thr Pro His Pro Pro Gln Lys Gln Ala
275 280 285
Tyr Gly Val Thr Leu Pro Thr Ser Val Leu Phe Ile Ala Gly His Asp
290 295 300
Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu Glu Leu Asn Trp Thr Leu
305 310 315 320
Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Glu Leu Val Phe Glu
325 330 335
Arg Trp Arg Arg Leu Ser Asp Asn Ser Gln Trp Ile Gln Val Ser Leu
340 345 350
Val Phe Gln Thr Leu Gln Gln Met Arg Asp Lys Thr Pro Leu Ser Leu
355 360 365
Asn Thr Pro Pro Gly Glu Val Lys Leu Thr Leu Ala Gly Cys Glu Glu
370 375 380
Arg Asn Ala Gln Gly Met Cys Ser Leu Ala Gly Phe Thr Gln Ile Val
385 390 395 400
Asn Glu Ala Arg Ile Pro Ala Cys Ser Leu
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<210> 2
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<212> DNA
<213>Escherichia coli (Escherichia coli)
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cccgtcaagc tgggctggct gacgccccgc ggcggcgagc tgattgccta cctgggccac 180
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ggccaggtgg ccattattgc cgacgtggac gagcgcacgc gaaagacggg cgaggccttc 300
gccgccggcc tggcccccga ctgcgccatt accgtgcaca cgcaggccga cacgtcgtcg 360
cccgaccccc tgttcaaccc cctcaagacg ggcgtgtgcc agctcgacaa cgccaacgtg 420
acggacgcca ttctgtcgcg cgccggcggc agcattgccg acttcacggg ccaccgacag 480
accgccttcc gagagctgga gcgcgtcctg aacttccccc agtccaacct gtgcctcaac 540
cgagagaagc aggacgagtc gtgctcgctg acccaggccc tgccctcgga attgaaagtg 600
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caccagtgga acaccctcct ctccctgcac aacgcccagt tctacctgtt acaacgaacg 780
cccgaggtgg cccgatcccg cgccacgccc ctcctggacc tcatcatggc cgccctcacg 840
ccccaccccc cccagaagca ggcctacggc gtgacgctcc ccacgtcggt gctcttcatt 900
gccggccacg acaccaacct cgccaacctg ggcggcgccc tagaactgaa ctggaccctc 960
cccggccagc ccgacaacac gccccccggc ggcgagctgg tcttcgagcg atggcgacga 1020
ctgtcggaca actcgcagtg gattcaggtc agcctggtgt tccagaccct ccagcagatg 1080
cgagacaaga cgcccctctc gctgaacacg ccccccggcg aggtcaagct caccctggcc 1140
ggctgcgaag agcgaaacgc ccagggcatg tgctcgctcg ccggcttcac ccagattgtg 1200
aacgaggccc gaattcccgc ctgctcgctc taa 1233
Claims (8)
1. a kind of trichoderma reesei engineered strain, which is characterized in that the trichoderma reesei engineered strain is carried for recombinating table
Up to the recombinant plasmid of phytase.
2. trichoderma reesei engineered strain as described in claim 1, which is characterized in that the amino acid sequence of the phytase is
SEQ ID NO:1。
3. trichoderma reesei engineered strain as claimed in claim 1 or 2, which is characterized in that the phytase, encoding nucleoside
Acid sequence is SEQ ID NO:2.
4. a kind of trichoderma reesei mutant strain, which is characterized in that the trichoderma reesei mutant strain is to described in claim 1
Trichoderma reesei engineered strain carry out ultraviolet mutagenesis after screening obtain.
5. trichoderma reesei mutant strain as claimed in claim 4, which is characterized in that the guarantor of the trichoderma reesei mutant strain
Hiding number is CCTCC NO:M2019405.
6. application of the trichoderma reesei engineered strain described in claim 1 in production phytase.
7. application of the trichoderma reesei mutant strain as claimed in claim 4 in production phytase.
8. a kind of method for producing phytase, which is characterized in that the method is using bacterial strain described in claim 1 or 4
Fermenting and producing phytase.
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